The molecular mechanisms involved in DNA replication are not yet fully understood because a well defined in vitro system capable of carrying out the entire replication process of double-standard DNA molecules has not been available. We have recently assembled the purified replication proteins coded by phage T4 into in vitro apparatuses capable of efficient replication of double-stranded DNA molecules by a process that is semi-conservative, discontinuous, faithful and generates product molecules lacking rapidly renaturable hairpin areas. It is planned to use this system composed of 6 purified proteins and pure DNA molecules to approach some of the unanswered questions in replication. We plan to analyze the factors involved in generation of the very high fidelity seen in DNA duplication. Using this system we also plan to determine the features in DNA templates that are recognized by the proteins to initiate DNA synthesis. In vivo, DNA exists in a tightly folded complex with proteins and the cell membrane. Using gentle lysis conditions, the intracellular T4-DNA can be isolated as compact DNA-protein complexes that serve as templates for in vitro synthesis. This synthesis is further stimulated by the addition of the purified replication proteins. Using this more natural template, we plan to study the roles played by the various accessory replication proteins coded by the phage (the products of DNA-delay and DNA arrest genes, some of which are membrane components). It is hoped that these studies will not only lead to clearer understanding of the mechanism of DNA-replication in prokaryotic cells but will also help us find proteins carrying out similar roles in eukaryotic cells.